异槲皮苷调节AMPK-TORC2磷酸化影响肝细胞糖异生的研究
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摘要
目的观察异槲皮苷对肝细胞糖异生及关键酶的影响,并探讨其影响肝细胞糖异生的机制。方法体外培养小鼠原代肝细胞,以乳酸和丙酮酸为糖异生底物,予异槲皮苷干预、以二甲双胍阳性为对照,分为空白对照组(Con)、糖异生诱导组(GN)、40μmol/L异槲皮苷组(40IQ)、80μmol/L异槲皮苷组(80IQ)、500μmol/L二甲双胍组(Met)。GOD法测定上清葡萄糖浓度,Western blot检测总AMPKα及AMPKαThr172磷酸化、TORC2蛋白表达及Ser171磷酸化,RT-PCR检测磷酸烯醇式丙酮酸羧激酶(PEPCK)、AMP活化的蛋白激酶(AMPK)、环腺苷酸诱导因子结合蛋白辅激活物2(TORC2)的mRNA表达水平。经AMPK抑制剂二氢脱氧吗啡预处理,观察上述条件下葡萄糖浓度、PEPCK、葡萄糖6磷酸酶(G6Pase)表达、AMPKα、TORC2蛋白水平及其磷酸化改变。结果 80IQ组上清葡萄糖浓度较GN组降低[(124. 62±24. 33)vs(179. 50±31. 86)μmol/L,P<0. 01]。GN组PEPCK、G6Pase表达高于Con组和80IQ组。Western blot检测结果显示,异槲皮苷增加pThr172-AMPKα和p Ser171-TORC2蛋白水平。经二氢脱氧吗啡预处理后,80IQ组、Met组葡萄糖浓度增加[(124. 62±24. 33)vs(168. 62±23. 71)μmol/L,(111. 50±25. 42)vs(138. 33±17. 58)μmol/L,P<0. 01]、PEPCK mRNA表达上调。异槲皮苷组抗磷酸化AMPKα(pT172-AMPKα)和抗磷酸化TORC2(p Ser171-TORC2)蛋白表达受到抑制。结论异槲皮苷抑制体外培养的小鼠原代肝细胞糖异生及关键酶基因转录,可能通过调节AMPK与TORC2磷酸化影响肝细胞糖异生。
Objective To observe the effect and mechanism of Isoquercitrin on hepatocyte glycogenesis and key enzymes,and to explore the mechanism of its effect on hepatocyte glycogenesis.Methods The primary hepatocytes of mice were cultured in vitro. Lactic acid and pyruvic acid were used as gluconeogenic substrates. The mice were divided into blank control group(Con),gluconeogenesis induction group(GN),40 μmol/L Isoquercitrin intervention group(40 IQ),80 μmol/L Isoquercetin intervention group(80 IQ)and 500 μmol/Lmetformin positive control group(Met). Glucose concentration in supernatant was determined by GOD method. The levels of total AMPKα and AMPKα Thr172 phosphorylation,TORC2 protein level and Ser171 phosphorylation were detected by Western blot. The m RNA expression levels of phosphoenolpyruvate car-boxykinase(PEPCK),AMP-activated protein kinase(AMPK)and cyclic adenylate inducer binding protein coactivator 2(TORC2)were detected by RT-PCR. After pretreatment with AMPK inhibitor dihydrodeoxy-morphine,changes in these indicators were observed under the above conditions.Results The concentration ofsupernatantglucosein 80 IQgroupwaslowerthanthatinGNgroup[(124. 62±24. 33)vs(179. 50±31. 86)μmol/L,P<0. 01]. The expressions of PEPCK and G6 Pase in GN group were higher than those in Con group and 80 IQ group. Western blot showed that Isoquercitrin increased the levels of p Thr172-AMPK alpha and p Ser171-TORC2 proteins. After pretreatment with AMPK inhibitor,the glucose concentration increased [(124. 62±24. 33)vs(168. 62±23. 71)μmol/L,(111. 50±25. 42)vs(138. 33±17. 58)μmol/L,P<0. 01] and the expression of PEPCK gene increased in 80 IQ and Met groups. In Isoquercetin group,the levels of anti-phosphorylated AMPKα(p T172-AMPKα)and anti-phosphorylated TORC2(p Ser171-TORC2)protein were significantly inhibited.Conclusion Isoquercitrin inhibits glycogenesis and transcription of key enzymes in primary cultured mouse hepatocytes,possibly by regulating the phosphorylation of AMPK and TORC2.
Objective To observe the effect and mechanism of Isoquercitrin on hepatocyte glycogenesis and key enzymes,and to explore the mechanism of its effect on hepatocyte glycogenesis.Methods The primary hepatocytes of mice were cultured in vitro. Lactic acid and pyruvic acid were used as gluconeogenic substrates. The mice were divided into blank control group(Con),gluconeogenesis induction group(GN),40 μmol/L Isoquercitrin intervention group(40 IQ),80 μmol/L Isoquercetin intervention group(80 IQ)and 500 μmol/Lmetformin positive control group(Met). Glucose concentration in supernatant was determined by GOD method. The levels of total AMPKα and AMPKα Thr172 phosphorylation,TORC2 protein level and Ser171 phosphorylation were detected by Western blot. The m RNA expression levels of phosphoenolpyruvate car-boxykinase(PEPCK),AMP-activated protein kinase(AMPK)and cyclic adenylate inducer binding protein coactivator 2(TORC2)were detected by RT-PCR. After pretreatment with AMPK inhibitor dihydrodeoxy-morphine,changes in these indicators were observed under the above conditions.Results The concentration ofsupernatantglucosein 80 IQgroupwaslowerthanthatinGNgroup[(124. 62±24. 33)vs(179. 50±31. 86)μmol/L,P<0. 01]. The expressions of PEPCK and G6 Pase in GN group were higher than those in Con group and 80 IQ group. Western blot showed that Isoquercitrin increased the levels of p Thr172-AMPK alpha and p Ser171-TORC2 proteins. After pretreatment with AMPK inhibitor,the glucose concentration increased [(124. 62±24. 33)vs(168. 62±23. 71)μmol/L,(111. 50±25. 42)vs(138. 33±17. 58)μmol/L,P<0. 01] and the expression of PEPCK gene increased in 80 IQ and Met groups. In Isoquercetin group,the levels of anti-phosphorylated AMPKα(p T172-AMPKα)and anti-phosphorylated TORC2(p Ser171-TORC2)protein were significantly inhibited.Conclusion Isoquercitrin inhibits glycogenesis and transcription of key enzymes in primary cultured mouse hepatocytes,possibly by regulating the phosphorylation of AMPK and TORC2.
引文
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[16]Jiang J,Dong H,Li B,et al.Berberine inhibits hepatic gluconeogenesis via the LKB1-AMPK-TORC2 signaling pathway in streptozotocin-induced diabetic rats.World JGastroenterol,2015,21:7777-7785.
[17]Akingbemi BT.Adiponectin receptors in energy homeostasis and obesity pathogenesis.Prog Mol Biol Transl Sci,2013,114:317-342.--
[2]陈琳,陆明,曹玉莉,等,黄秋葵多糖调节PEPCK和AMPK表达抑制高脂饮食小鼠肝糖异生.疑难病杂志,2017,16:287-292.
[3]王英锋,王琰,高效离心分配色谱法分离制备秋葵种子中的异槲皮苷.药物分析杂志,2012,32:1981-1984.
[4]王英锋,王琰,秋葵荚中两种黄酮苷的分离鉴定及含量测定.首都师范大学学报(自然科学版),2012,3:22-24.
[5]Huang L,He Y,Ji L,et al.Hepatoprotective potential of isoquercitrin against type 2 diabetes-induced hepatic injury in rats.Oncotarget,2017,8:101545-101559.
[6]Xu Y,Wang L,He J,et al.Prevalence and control of diabetes in Chinese adults.JAMA,2013,310:949-958.
[7]Phuwamongkolwiwat P,Hira T,Hara H.A nondigestible saccharide,fructooligosaccharide,increases the promotive of a flavonoid,α-glucosyl-isoquercitrin,on glucagon-like peptide 1(GLP-1)secretion in rat intestine and enteroendocrine cells.Mol Nutr Food Res,2014,58:1581-1584.
[8]Kim S,Kang M,Jin Y,et al.Antioxidant activities and polyphenol content of Morus alba leaf extracts collected from varying regions.Biomed Rep,2014,2:675-680.
[9]Cai D,Su L,Guo S,et al.Effect of flavonoids from Abelmoschus manihot on proliferation,differentiation of 3T3-L1preadipocyte and insulin resistance.China J Chin Mater Med,2016,41:4635-4641.
[10]Yabaluri N,Bashyam D.Hormonal regulation of gluconeogenic gene transcription in the liver.J Biosci,2010,35:473-484.
[11]An H,He L.Current understanding of metformin effect on the control of hyperglycemia in diabetes.J Endocrinol,2016,228:R97-R106.
[12]Rena G,Pearson R,Sakamoto K.Molecular mechanism of action of metformin:Old or new insights?.Diabetologia,2013,56:1898-1906.
[13]Shaw J,Lamia A.The kinase LKB1 mediateds glucose homeostasis in liver and therapeutic effects of metformin.Science,2005,310:1642-1646.
[14]Deidre J,Andy N,Accalia F,et al.Glucose controls CREBactivity in islet cells via regulated phosphorylation of TORC2.PNAS,2008,105:10161-10166.
[15]Yoon S,Lee W,Ryu D,et a1.Suppressor of MEK null(SMEK),proteinphosphatase 4 catalytic subunit(PP4C)is a key regulator of hepatic gluconeogenesis.Proc Nad Acad Sci USA,2010,107:17704-17709.
[16]Jiang J,Dong H,Li B,et al.Berberine inhibits hepatic gluconeogenesis via the LKB1-AMPK-TORC2 signaling pathway in streptozotocin-induced diabetic rats.World JGastroenterol,2015,21:7777-7785.
[17]Akingbemi BT.Adiponectin receptors in energy homeostasis and obesity pathogenesis.Prog Mol Biol Transl Sci,2013,114:317-342.--